JP2000275541A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To correct the Z-directional luminance variation of a scanning type laser microscope. SOLUTION: A signal processing part 110 which processes an obtained image is equipped with a mean luminance calculation part 121 which calculates the mean luminance of respective obtained image data, a correction coefficient calculation part 122 which calculates correction coefficients for reference image data selected out of the obtained image data from the mean luminance of the image data calculated by the calculation part 121, and a luminance conversion part 123 which corrects the luminance according to the correction coefficients calculated respectively for the image data.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser microscope and, more particularly, to a laser microscope for correcting a medium difference in a Z direction of a sample.
The present invention relates to a laser microscope that corrects luminance in a depth direction of a three-dimensional image and displays an image that is easy for an observer to see.

[0002]

2. Description of the Related Art An optical microscope has a configuration in which a sample on a slide mounted on a stage is magnified and observed with an objective lens. Generally, an illumination system for the sample uses a condenser lens to illuminate light from a light source such as a lamp. In this case, a structure was employed in which the sample was used so as to be even over the entire observation area of the sample.

[0003] However, when such a structure is adopted as an illumination system, there are problems such as flare and the like, and it is very difficult to observe a sample with low contrast. As a solution to such a problem, a scanning optical microscope which is a point light projection type (spot light projection type) optical microscope has been proposed.

A scanning optical microscope irradiates a sample to be observed with light from a point light source via an objective lens in the form of a dot, thereby transmitting light (transmitted light), reflected light, or point light. The fluorescent light generated from the sample by irradiating the light in the form of a spot is imaged again as a dot through an optical system such as an objective lens, and this is detected by a detector through a pinhole to obtain the density information of the image. It is something that you get.

[0005] However, only this can obtain only one point of light and shade information in which light emitted from a point light source becomes a point. Therefore, the light from the point light source moves the stage in the X-axis and Y-axis directions while the optical axis is fixed, or the light from the point light source is applied to the sample on the stage in the X-axis and Y-axis directions. By using a scanning mechanism that deflects the light to 2
Dimensionally scanned. Shading information obtained from light from the sample is displayed on an image display device such as a CRT display in synchronization with two-dimensional scanning on the sample, so that a two-dimensional image of the sample can be observed. The above are the principle components of the scanning optical microscope.

It is widely known that the use of laser light as a point light source improves the resolution of an image, and is called a scanning laser microscope. Scanning laser microscopes use transmitted light (reflected light) from a sample being laser-scanned.
Is usually converted into an electric signal by photoelectric conversion by a photomultiplier tube or a photodiode as a detector, and the converted signal is stored, processed, and displayed as image data.

As a general example, a basic configuration of a conventional scanning laser microscope will be described with reference to FIG. Reference numeral 101 denotes an optical microscope main body.
A spot light is generated on the surface of the sample on the microscope stage provided in 1. Actually, since XY scanning is performed on the sample surface, the laser light guided into the optical microscope 101 is 2
In the dimensional scanning mechanism 103, the optical path of the spot light with respect to the objective lens is shifted to the XY optical path.

As a result of irradiating the sample with spot light,
The information of the reflected light or the fluorescence from the sample is passed through the objective lens, and the pinhole plate 10 is passed through the two-dimensional scanning mechanism 103.
4, the light is received by the light detection unit 105 and photoelectrically converted into an electric signal. The pinhole plate 104 is provided with a pinhole having a predetermined diameter, and is disposed at an image forming position on the front surface of the light detection unit 105. Light passing through the pinhole plate 104 is only information focused at an observation point on the sample surface. Can be detected, and a confocal effect can be obtained. still,
To drive the two-dimensional scanning mechanism unit 103, a two-dimensional scanning driving unit 119 generates a scanning control signal, and the signal processing unit 110 performs data processing based on the signal from the scanning control signal.

The detected electric signal is sent to a signal processing unit 110.
Is processed and displayed on the display unit 131. First, a desired signal is amplified by the gain variable unit 111, and then the desired signal is increased or decreased by the offset adjustment unit 112. The set amount is set to a desired value in the D / A converters 117 and 118 by the CPU 116, respectively. Next, the signal output from the offset adjusting unit 112 is output to the A / D converter 113.
After the analog / digital conversion, the image data is temporarily stored in the storage unit 114 as image data.

[0010] The stored image data is thereafter processed, displayed, and stored. Processing refers to performing desired image processing by the CPU 116, and displaying refers to outputting image data from the storage unit 114, displaying the image data on the display unit 131 through the D / A converter 115, and observing the image. To be able to

When the depth direction, that is, three-dimensional information is required, the Z-scan driver 120 is moved to a desired Z position, and necessary images are sequentially constructed in the storage unit 114.
Thereby, display and observation of a three-dimensional image are also possible.

As described above, the laser microscope can handle three-dimensional image observation of biological and metal three-dimensional samples, and other measurements. An application example of measurement of a three-dimensional sample using a laser microscope is disclosed in JP-A-6-265317. This is an example in which the thickness of a sample is measured based on laser reflected light from a reflective sample.

[0013]

Generally, when a three-dimensional stereoscopic image of an organism or the like is constructed using a laser microscope, the constructed three-dimensional image has a difference in fluorescence luminance depending on the observation depth. For example, as shown in FIG. 13, when observing a thick and homogeneous observation sample (sample) 1301, a position where the distance from the objective lens 1302 to the sample 1301 is short is referred to as a shallow observation position and is referred to as an A surface. . Further, a place where the distance is far is referred to as a deep observation position and is referred to as a B surface. The luminance on the B side is lower than the luminance on the A side. This is because incident light blocking and reflected light (transmitted light) is lost in the sample due to reflection, scattering, and absorption on the B surface, as compared with the laser light and fluorescence at the position of the A surface.

Therefore, when a three-dimensional image is constructed from a plurality of slice images obtained, a phenomenon appears in which the luminance of the image on the XY plane becomes lower as the Z axis becomes deeper.
As described above, depending on the sample, the result of constructing a three-dimensional image of a biological sample usually does not mean that the brightness is uniform in the Z direction. The brightness decreases as the observation depth increases, and the entire image in the Z direction is visually observed. An image that is hard to grasp and hard to see is displayed, which hinders observation. Therefore, in order to make the image more easily viewable, it is desirable that the brightness can be converted and displayed so that the brightness in the Z direction can be easily viewed.

However, in general, reflected light (transmitted light) from the inside of a sample is largely affected by light absorption and scattering in the sample, and the luminance change in the Z direction depends on the observation sample and the reagent.
Therefore, there is a problem that it is difficult to determine a change in light in the depth direction of the fluorescence.

Japanese Patent Application Laid-Open No. 6-265317 discloses an observation method capable of measuring the thickness of a metal reflection sample in the depth direction, but cannot be applied to a fluorescent observation biological sample. Accordingly, the brightness in the Z direction cannot be corrected in the configuration of the conventional example.

An object of the present invention is to provide a laser microscope which can correct a change in luminance according to the observation depth of a sample and can easily observe the sample.

[0018]

Means for Solving the Problems [Configuration] The present invention is configured as follows to achieve the above object.

(1) A laser microscope according to the present invention (claim 1) includes a laser light source for emitting a laser beam to a sample, and an optical system for irradiating the laser beam to a focal position of the sample. Detecting means for detecting light from the focal position of the sample, output means for outputting data detected by the detecting means, and means for changing the focal position of the laser light relative to the sample And a means for correcting the luminance of each data obtained for each focal position.

(2) The laser microscope according to the present invention (claim 2) detects the reflected light or the transmitted light from the sample while scanning the spot light with respect to the two-dimensional plane in the XY direction of the sample. In a laser microscope that obtains image data of a two-dimensional plane in the direction and changes the focal position of the spot light in the Z direction to obtain the image data of a plurality of different observation positions, an average of the obtained image data is obtained. From the average luminance calculating means for calculating the luminance, and from the average luminance of the image data calculated by the average luminance calculating means,
A means for calculating a correction coefficient for reference image data selected from a plurality of acquired image data; and a means for performing luminance correction based on the correction coefficient calculated for each image data. It is characterized by becoming.

(3) The laser microscope of the present invention (claim 3) reflects a spot light on a two-dimensional plane in the XY directions of a sample containing a fluorescent sample stained with a fluorescent substance while reflecting the sample from the sample. A laser microscope that acquires image data of a two-dimensional plane in the XY directions by detecting light or transmitted light, and acquires the image data at a plurality of different observation positions by changing the focal position of the spot light in the Z direction. A means for extracting a fluorescent part from the image data, an average luminance calculating means for calculating an average luminance of the extracted fluorescent part, and a plurality of images obtained from the average luminance calculated by the average luminance calculating means. Means for calculating a correction coefficient for the reference image data selected from the data, and brightness correction based on the correction coefficients calculated for each image data Characterized by comprising comprises a means for performing.

(4) The laser microscope according to the present invention (claim 4) scans the sample by changing the focal position of the spot light, and obtains a plurality of different XY planes from reflected light or transmitted light from the sample. In the laser microscope for acquiring image data in the image data, the spot light and the reflected light or the transmitted light are attenuated with respect to the image data in accordance with the information on the focal position where the image data is acquired. It is characterized by comprising means for correcting a change in luminance.

Preferred embodiments of the present invention are shown below.

There is provided means for displaying a three-dimensional image or based on the corrected image data.

A photoelectric conversion means for detecting the reflected light or the transmitted light to obtain an electric signal, a means for adjusting a gain of the converted electric signal, a means for performing an offset adjustment,
It comprises means for converting the signal from analog to digital, means for storing an image, and means for moving the sample in the Z direction (depth).

While scanning the spot light with respect to the two-dimensional plane in the XY direction of the sample and detecting the reflected light or transmitted light from the sample, image data of the two-dimensional plane in the XY direction is obtained. A laser microscope that obtains the image data at a plurality of different observation positions by changing the focal position of the spot in the Z direction. And means for correcting a change in luminance of the image data due to attenuation of light and the reflected light or transmitted light.

[Operation] The present invention has the following operation / effect by the above configuration.

In the first configuration, the luminance of each data obtained for each focal position is corrected, so that the difference in the medium in the Z direction can be corrected, and the Z direction can be compared with the image before correction. Can be eliminated.

In the second configuration, the average luminance of the image data is calculated, and a correction coefficient for the average luminance of the reference image data is calculated from the average luminance to correct the image data. Alternatively, the difference in the medium in the Z direction can be corrected without delaying the processing time from the data acquisition of the XZ image to the display, and there is no difference in luminance in the Z direction compared to the image before correction, and in the depth direction XZ, YZ, and XYZ images that can be easily observed are obtained.

Further, by extracting the fluorescent portion from the image data, the influence of the background upon calculating the average value can be reduced, and a brightness-converted image that can be easily viewed can be realized. Further, since the threshold value for separating the fluorescent image portion and the background portion can be adjusted, an optimal final image suitable for the sample at the time of observation can be displayed.

In the fourth configuration, an image having dark luminance in the Z direction can be converted into an easy-to-view image by performing luminance conversion in accordance with a natural phenomenon, and XZ, YZ, and XYZ images that can be easily observed in the depth direction can be obtained.

[0032]

Embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment] FIG. 1 is a block diagram showing a schematic configuration of a scanning laser microscope according to a first embodiment of the present invention.

Reference numeral 101 denotes an optical microscope main body, and a laser light source 1
The laser light is directly guided from the 02 to the two-dimensional scanning mechanism 103 without using a fiber or a fiber, and the guided laser light is irradiated on the sample (on the focal plane) on the microscope stage via the objective lens. I do. Note that the scanning control signal generated by the two-dimensional scanning drive control unit 119
By driving the two-dimensional scanning mechanism 103, the sample is scanned XY with the spot light.

The XY scanning of the spot light on the sample is performed by using two galvanometer mirrors (X galvanometer mirror 201 and Y galvanometer mirror 202) as shown in FIG. Scan in the direction and the Y direction. The signals necessary for the scanning in the XY directions are referred to as X, Y or horizontal (H) and vertical (V) synchronization signals. As a result, the spot light is scanned XY on the sample surface.

As a result of irradiating the spot light, light information from the sample, for example, fluorescence information, passes through the objective lens, passes through the two-dimensional scanning mechanism 103 and the pinhole plate 104, Is photoelectrically converted to Note that a photomultiplier tube (PMT) is often used as a photoelectric conversion element of the light detection unit 105.

The pinhole plate 104 is a plate in which a pinhole having a predetermined diameter is formed, and is disposed at an image forming position on the front surface of the light detection unit 105. From the light that has passed through the pinhole plate 104, only information at one point that has been focused at the observation point on the sample surface can be detected, and a confocal effect can be obtained.

The detected electric signal is sent to a signal processing unit 110.
And is displayed on the display unit 131. Signal processing unit 1
The signal processing in 10 will be described. First, a desired signal amplification is performed by the gain variable section 111, and then, an increase or decrease of the signal is set by the offset adjusting section 112 so as to have a desired signal amount. The set values of the signal amplification amount and the offset amount are
The D / A converters 117 and 1 are sent from the CPU 116, respectively.
18 for each set value.

Next, the signal output from the offset adjusting unit 112 is subjected to analog / digital conversion by the A / D converter 113 and then stored in the storage unit 114. Normally, when an XY image is acquired, the average luminance calculation unit 121 does not perform any processing, and the D / A converter 115 performs digital / analog conversion, and the converted signal is displayed from the signal processing unit 110 as output means. Part 131 is transmitted,
A two-dimensional image is displayed.

On the other hand, when it is desired to acquire a three-dimensional image in the XYZ directions, the position (height) Z of the microscope stage is moved in desired steps by the Z scanning drive unit 120, and an XY image is acquired for each observation position. Finally, a three-dimensional image is constructed. The constructed three-dimensional image is stored in the storage unit 114.

When the three-dimensional image is displayed as it is, the image is not subjected to the luminance conversion in the Z direction. Therefore, the average luminance calculation unit 121, the correction coefficient calculation unit 122, and the luminance conversion unit 1
At 23, luminance conversion in the Z direction is performed, and display is performed as new image data.

In the brightness conversion method in the Z direction, brightness conversion is performed so as to compensate for light attenuation and scattering at a position deep in Z, as a general tendency, so that display can be performed. Hereinafter, the method will be described. When acquiring a three-dimensional image, determining the observation position Z _n of the end take the observation position Z ₁ of the beginning of the sample taken. Normal,
The microscope stage is driven in a direction in which the sample moves away from the objective lens. Therefore, when the observation position of the first take and Z _1, the observation position is bright two-dimensional image so shallow position. First, to obtain the two-dimensional image of the observation position Z ₁ first.

Next, the obtained observation position Z₁2D drawing in
The average brightness of the entire screen of the image is calculated by the average brightness calculation unit 121
And place it in the observation position Z₁Average brightness I at₁And Observation
Position Z ₁Average brightness I at₁To the standard (after correction)
The average brightness value is used.

Next, the microscope stage is moved to the next observation position Z.
Driven to _2, to obtain the XY image at the observation position Z _2. Then, the average luminance I of the XY image at the acquired observation position Z ₂ is obtained.
₂ is calculated (FIG. 3). The average luminance I ₂ at the observation position Z ₂
Becomes the value of the average luminance I ₁ at the observation position Z ₁ ,
The correction coefficient is obtained by the correction coefficient calculation unit 122. By the correction coefficient calculation unit 122, the correction coefficient k ₂ of the observation position Z ₂ is from the average luminance I ₂ at the observation position Z ₁ as a reference, is calculated as _{k 2 = I 1 / I 2} (1) .

Then, the luminance conversion section 123 performs luminance correction by multiplying the image at the observation position Z ₂ by a correction coefficient k ₂ (FIG. 4). Then, the correction Similarly, the 2-dimensional image in the following other viewing position Z _i. Thereby, the brightness conversion of the two-dimensional image obtained each time the observation position Z is moved can be performed without delaying the processing time from data acquisition to display.

For the cross-sectional image in the XZ (YZ) direction, the average luminance value is calculated for the pixels in the line in the X (Y) direction from the two-dimensional image as the average luminance value. To go. The image area for calculating the average luminance is not limited to the entire screen, but may be an arbitrary area including the center of the image in some cases.

According to this embodiment, the XYZ image or the YZ
In order to correct the difference in the medium in the Z direction without delaying the processing time from the acquisition of the image or XZ image data to the display,
The luminance conversion is performed so as to eliminate the luminance difference in the Z direction as compared with the image before correction, and XZ, Y that can be easily observed also in the depth direction
Z, XYZ images are obtained.

[Second Embodiment] FIG. 5 is a block diagram showing a schematic configuration of a scanning laser microscope according to a second embodiment of the present invention. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted.

In the first embodiment, the average luminance value is calculated for the entire screen of the XY image, all the pixels of the X line, or all the images of the Y line. However, when a sample containing a fluorescent sample stained with a fluorescent substance is generally observed, FIG.
As shown in (2), for example, on an XY image, there are a portion of cells (fluorescent sample) that emits fluorescence and a background portion without fluorescence.
Therefore, it is desirable that the image to be calculated when performing the luminance conversion be an image of only the fluorescent portion, and it is not preferable to calculate the image including the background portion without the fluorescent portion.

If the calculation is performed with the background portion included, the average luminance value becomes low. Therefore, in the image after the luminance correction,
An image in which the fluorescent portion is brighter than the reference average luminance value I ₁ is obtained. Therefore, in the case of the first embodiment, if a background portion is included in the calculation of the average value, an image desired by the observer may not be obtained.

Therefore, in the present embodiment, the drawback of the first embodiment is compensated for by providing the fluorescent portion extracting section 124 for extracting the fluorescent portion from the XY image and using the average luminance value of only the fluorescent image not including the background. be able to.

More specifically, only the acquired XY image exceeding the luminance level set in advance by the fluorescent part extracting unit 124 is determined as the fluorescent image data, and the average luminance value is calculated. Since the observer can set the luminance level as desired by an external input device or the like (not shown), an appropriate value can be determined as the threshold value by checking the actual image of the sample or the state of the electrical noise of the device. Subsequent processing is the same for the XYZ, XZ, and YZ images as in the first embodiment, and a description thereof will be omitted.

According to the configuration of the present embodiment, the influence of the background when calculating the average value is reduced, and an easy-to-view luminance-converted image can be realized. Further, since the threshold value for separating the fluorescent image portion and the background portion can be adjusted, an optimal final image suitable for the sample at the time of observation can be displayed.

[Third Embodiment] FIG. 6 is a block diagram showing a schematic configuration of a scanning laser microscope according to a third embodiment of the present invention. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted.

In the present embodiment, the observer performs luminance conversion in the Z direction using the acquired XYZ image while viewing the XZ or YZ image. From the obtained XYZ image, XZ, YZ
An XZ or YZ section in which the brightness of the cells is distributed in the Z direction is cut out by the cutout section 125, and a brightness distribution in the Z direction is obtained for the cells on the cross section.

For example, as shown in FIG.
_1, calculates the luminance distribution with respect to the line of the deep observation position Z ₂ (FIG. 8). Using this brightness change, obtain a correction coefficient for correcting the luminance of the other viewing positions Z _i bright luminance of observation position Z ₁ as in the first embodiment performs correction for each observation position. Thereby, it is possible to perform the luminance conversion at a desired observation position of the observer compared to the case where the background of the first embodiment is not considered.

According to the configuration of this embodiment, XZ or YZ
Since the luminance distribution in the depth direction is calculated based on the conversion data from the cut-out image, a good image can be obtained after the correction without being affected by the background image.

[Fourth Embodiment] In this embodiment, an example in which luminance correction is performed using an approximate expression will be described. It is assumed that the condition is a homogeneous sample and that the fading of the fluorescence can be ignored. Under such conditions, the increase or decrease of light in the sample is considered as follows. In general, when light travels through a substance (medium), assuming that the original light intensity is I, an attenuation coefficient G of the light intensity in the substance is expressed as a rate of change when the light travels a minute distance dz: − (DI / I) (1 / dz) (2)

Therefore, when the intensity of the laser beam immediately before entering the substance is I ₀ , the light intensity I _(Z) at the observation position Z is I _(Z) = I ₀ × exp (−GZ) (3) It can be seen that the laser light is absorbed and attenuated in the sample. That is, the intensity of the laser light in the sample decreases exponentially as it goes deeper into the sample (FIG. 9).

The same concept can be applied to the fluorescence intensity emitted by the reagent (fluorescent substance), and the light intensity is attenuated from the time when the fluorescence is emitted until the light reaches the detector. Assuming that the amount of fluorescent light generated from a certain observation position Z is F ₀ and the attenuation coefficient B at that time is, the amount of fluorescent light F in which the fluorescent light passing through the substance is detected by the detector is expressed as a function of _Z : F _(Z) = F ₀ × exp (−BZ) (4)

The amount of fluorescence F ₀ is determined by the intensity I of the laser beam.
Assuming that the spontaneous emission coefficient of the reagent (fluorescent substance) is A ₀ in proportion to _(Z) , F ₀ = A ₀ × I ₀ × exp (−GZ) (5) Therefore, when the observation position is at the depth Z, the amount of fluorescence F _(Z) detected by the light detection unit is as follows: F _(Z) = A ₀ × I _X × F ₀ × exp (− (B + G) Z) (6) ).

Therefore, the amount of fluorescent light detected by the light detecting section decreases exponentially as a deep portion of the sample is observed. As described above, since the light intensity changes according to the observation depth Z, the detected fluorescence luminance can be applied to the expression (6) (FIG. 10).

Assuming that the fluorescence intensity F _(z) is the average luminance, the total initial value (A ₀ × I ₀ ×
Assuming that F ₀ ) is C ₀ and the attenuation coefficient (B + G) is k (FIG. 1).
1), F = C ₀ × exp (−kZ) (7)

From the distribution of each Z position of the observed XYZ image and the average luminance, the coefficient C ₀ , k of the equation (7) with a small error is obtained by the least squares method. The value obtained in the first to third embodiments is used as the average luminance value at that time.

Then, the observed luminance value is corrected. The observed luminance is multiplied by a correction coefficient to perform luminance conversion in the Z direction, and a luminance detected when there is no attenuation of incident light and fluorescence in the sample is obtained. Then, the corrected luminance F _H is calculated as follows: F _H = F _z × C ₀ (1−exp [−kZ]) (8) where F _z is the luminance at each observation position Z.

These correction processes are performed by the CPU 116. Thus, for each observation position Z, the correction is performed by converting the luminance value of the XY image or the X line or the Y line. The corrected data is stored again in the storage unit 114 separately from the acquired image and displayed through the D / A converter 115.

Therefore, it is possible to obtain a luminance conversion image according to natural phenomena, instead of uniform luminance value adjustment. X
Similar processing can be performed on Z and YZ images for each X line.

As described above, these processes can be realized by hardware or software.

According to the structure of this embodiment, an image having dark luminance in the Z direction can be converted into an easy-to-view image by performing luminance conversion according to a natural phenomenon, and XZ, YZ, XYZ that can be easily observed in the depth direction.
An image is obtained. With the above-described method, by displaying an image which is easy to view after luminance conversion, a desired image of the operator and the observer can be provided.

The present invention is not limited to the above embodiment. For example, the invention is generally applicable to systems that can acquire XYZ images, for example, where the detector is P
A system using a detector such as a TV camera instead of the MT is also possible. In this case, there are no restrictions on the camera size and the signal system. In addition, these processes convert the input signal into
The present invention can be applied even when parallel processing is performed not only for one system but also for a plurality of inputs. Further, the photodetector 105 can efficiently perform photoelectric conversion of a photodiode (PD), a CCD, a CMD, etc., and is not limited to the PMT.

The two-dimensional scanning mechanism 103 includes a galvanometer mirror, a resonance galvanometer mirror, a polygon mirror,
OD may be used as long as XY scanning can be controlled.

The present invention can be variously modified and implemented without departing from the gist of the present invention.

[0073]

As described above, according to the present invention, a change in luminance according to the observation depth of a sample can be corrected, and the sample can be easily observed.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a schematic configuration of a scanning laser microscope according to a first embodiment.

FIG. 2 is a diagram showing XY scanning of laser light using a galvanomirror.

FIG. 3 is a diagram schematically illustrating a sample at each observation position.

FIG. 4 is a view for explaining the concept of luminance correction.

FIG. 5 is a block diagram showing a schematic configuration of a scanning laser microscope according to a second embodiment.

FIG. 6 is a block diagram showing a schematic configuration of a scanning laser microscope according to a third embodiment.

FIG. 7 is a diagram illustrating a correction method according to a third embodiment.

FIG. 8 is a diagram illustrating a correction method according to a third embodiment.

FIG. 9 is a diagram illustrating a correction method according to a fourth embodiment.

FIG. 10 is a diagram illustrating a correction method according to a fourth embodiment.

FIG. 11 is a view for explaining a correction method according to a fourth embodiment.

FIG. 12 is a block diagram showing a schematic configuration of a conventional scanning laser microscope.

FIG. 13 is a diagram illustrating a case where an observation sample (sample) is observed.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 101 ... Optical microscope 102 ... Laser light source 103 ... Two-dimensional scanning mechanism part 104 ... Pinhole plate 105 ... Photodetection part 110 ... Signal processing part 111 ... Gain variable part 112 ... Offset adjustment part 113 ... A / D converter 114 ... Storage part 115, 117, 118 D / A converter 116 CPU 119 Two-dimensional scan drive controller 120 Z scan drive 121 average luminance calculator 122 correction coefficient calculator 123 luminance converter 124 fluorescent part extractor 131 Display unit

Continued on the front page F-term (reference) 2G043 AA03 BA16 CA03 DA02 EA01 FA01 FA02 GA02 GA08 GA21 GB03 GB19 GB21 KA09 LA02 MA01 NA01 NA06 NA13 2G059 AA05 BB08 EE01 EE02 EE07 FF03 GG01 JJ11 JJ17 KK02 MM03 MM05 AC04 AC34 AF13 AF14 AF21 AF25

Claims (4)

[Claims] 1. A laser light source for emitting laser light to a sample, an optical system for irradiating the laser light to a focal position of the sample, and detecting light from the focal position of the sample. And a means for outputting data detected by the detecting means; and a means for relatively changing a focal position of the laser light with respect to the sample. A laser microscope comprising means for correcting the luminance of each data obtained every time. 2. An image data of a two-dimensional plane in the XY direction is obtained by detecting reflected light or transmitted light from the sample while scanning a spot light on the two-dimensional plane in the XY direction of the sample. In a laser microscope for acquiring the image data at a plurality of different observation positions by changing the focal position of the spot light in the Z direction, an average luminance calculating means for calculating an average luminance of each of the obtained image data; Means for calculating a correction coefficient for the reference image data selected from the plurality of image data obtained from the average luminance of the image data calculated by the calculation means; and a correction coefficient calculated for each image data. A means for performing luminance correction based on the laser microscope. 3. A method of detecting a reflected or transmitted light from a sample while scanning a spot light on a two-dimensional plane in the XY direction of a sample including a fluorescent sample stained with a fluorescent substance, thereby detecting a two-dimensional plane in the XY direction. A laser microscope that obtains image data of a two-dimensional plane, and obtains the image data of a plurality of different observation positions by changing the focal position of the spot light in the Z direction; and a means for extracting a fluorescent portion from the image data. Average luminance calculating means for calculating an average luminance of the extracted fluorescent portion; and a correction coefficient for reference image data selected from a plurality of image data obtained from the average luminance calculated by the average luminance calculating means. And a means for performing luminance correction based on the correction coefficient calculated for each image data. Laser microscope. 4. An image data of a two-dimensional plane in the XY direction is obtained by detecting reflected light or transmitted light from the sample while scanning a spot light on the two-dimensional plane in the XY direction of the sample. In a laser microscope that acquires the image data at a plurality of different observation positions by changing the focal position of the spot light in the Z direction, according to information on the focal position at which the image data is acquired,
A laser microscope comprising means for correcting a change in luminance of the image data due to attenuation of the spot light and the reflected light or transmitted light with respect to the image data.